Heparin binding motif and use thereof

ABSTRACT

A method for reducing cytotoxicity of eosinophil derived toxins comprising administering to a subject an effective amount of heparin, heparan sulfate, a potent heparanase inhibitor or a polypeptide which has sequence as follows: BZBXBX, XBBBXXBX, XBBXBX, BBXXBBBXXBB, BXBBXB, XBBBXXBBBXXBBX, or TXXBXXTBXXXTBB, wherein X represents any amino acid, Z represents an aromatic amino acid, and B represents a basic amino acid and T represents a turn.

FIELD OF THE INVENTION

The present invention relates to a heparin binding motif of eosinophiltoxins and use thereof.

BACKGROUND OF THE INVENTION

Eosinophil cationic protein (ECP), a member of the ribonuclease A (RNaseA) superfamily, is found in the specific granules of eosinophilicleukocytes. It is a single polypeptide with a molecular mass rangingfrom 16 to 21.4 kDa due to varying degrees of glycosylation. It shows a67% amino acid sequence identity with eosinophil-derived neurotoxin(EDN), another eosinophil-secreted RNase. Although ECP shares theoverall three-dimensional structure of RNase A, it has relatively lowerRNase activity (Boix, E., et al. (1999) Journal of Biological Chemistry274, 15605-15614). ECP released by activated eosinophils contributes tothe toxicity against helminth parasites, bacteria, and single-strand RNAviruses (Lehrer, R., et al. (1989) Journal of Immunology 142, 4428-4434;Domachowske, J. B., et al. (1998) Nucleic acids research 26, 3358-3363).Together with other proteins secreted from eosinophils such as EDN,eosinophil peroxidase (EPO; also EPX), and major basic protein (MBP),ECP is thought to cause damage to epithelial cells, a common feature ofairway inflammation in asthma (Gleich, G J. (2000) Journal of Allergyand Clinical Immunology 105, 651-663).

The mechanism underlying the cytotoxic property of ECP is unclear. Ithas been hypothesized that ECP cytotoxicity is due to destabilization oflipid membranes of target cells (Young, J., et al. (1986) Nature 321,613-616), and the degree of cytotoxicity is dependent on the cellularconcentration (Carreras, E., et al. (2005) Molecular and CellularBiochemistry 272, 1-7). The binding of ECP to target cells has beenattributed to its high arginine content (estimated pI=10.8), whichfacilitates the interaction between ECP and negatively charged moleculeson the cell surface (Carreras, E., et al. (2005) Molecular and CellularBiochemistry 272, 1-7; Carreras, E., et al. (2003) Biochemistry 42,6636-6644). Recently, we found that binding and endocytosis of ECP intobronchial epithelial cells were greatly dependent on the cell surfaceglycosaminoglycan (GAG), specifically heparan sulfate proteoglycans(HSPG) (Fan, T. C., et al. (2007) Traffic 8, 1778-1795). Thecytotoxicity of ECP was severely reduced toward cell lines with heparansulfate (HS) deficiency.

Heparin and HS are complex polysaccharides composed of alternating unitsof hexuronic acid and glucosamine. The uronic acid residues of heparintypically consist of 90% L-idopyranosyluronic acid and 10%D-glucopyranosyluronic acid (Capila, I. and Linhardt, R. J. (2002)Angewandte Chemie International Edition 41, 391-412). The N position ofglucosamine may be substituted with an acetyl or sulfate group. The 3-0and 6-0 positions of glucosamine and the 2-0 of uronic acid may besulfated. Through the combination of different negatively chargedmoieties, heparin and HS have been demonstrated to bind a variety ofproteins with diverse functions, including growth factors, thrombin,chemokines and viral proteins. The HS chains contain domains with a highlevel of sulfation and epimerization (S-domains), regions with mixedN-acetylation and N-sulfation (NA/S-domains), and unmodified domainsthat are mostly N-acetylated and contain little sulfate (Tumova, S., etal. (2000) The international journal of biochemistry & cell biology 32,269-288). Because HS chains contain heparin regions, heparin and itsmimetics can be used to study interactions between proteins andpolysaccharides.

The structure of ECP has been determined and refined to a resolution upto 1.75 Å, displaying a folding topology that involves three α helicesand five β strands (Mallorqui-Fernandez, G, et al. (2000) Journal ofMolecular Biology 300, 1297-1307). The most interesting feature is the19 surface-oriented arginine residues, conferring a strong basiccharacter to ECP. However, the heparin binding site in ECP has not beenidentified. Heparin binding domains within proteins usually contain ahigh proportion of positively charged residues, which bind to the acidicgroups of heparin through electrostatic interactions. It has beenproposed that the three-dimensional structure of the HS chain iscritical for protein binding (Hileman, R. E., et al. (1998) BioEssays20, 156-167). However, not much is known about the three-dimensionalstructure of HS. After examining a series of heparin-binding proteinsequences, Cardin and Weintraub proposed that the pattern XBBBXXBX orXBBXBX (where X represents hydrophobic or uncharged amino acids, and Brepresents basic amino acids) is responsible for HS binding to otherproteins (Cardin, A. D. and Weintraub, H. J. (1989) Arteriosclerosis(Dallas, Tex. 9, 21-32). In addition, the following sequences have alsobeen reported to serve as heparin binding motifs.

BBXXBBBXXBB (where B is a positively charge residue (arginine, lysine orhystidine) and X is any residue) (Olenina, L. V, et al. (2005) J ViralHepat 12, 584-593).

BXXBBXB (where B is a basic residue and X is any residue) (Wu, H. F., etal. (1995) Blood 85, 421-428).

XBBBXXBBBXXBBX (where B is a basic residue and X is any residue)(Andersson, E., et al. (2004) Eur J Biochem 271, 1219-1226; Sobel, M.,et al. (1992) The Journal of biological chemistry 267, 8857-8862).

TXXBXXTBXXXTBB (where B is a basic residue, X is any residue, and Tdefines a turn) (Capila, I. and Linhardt, R. J. (2002) Angewandte ChemieInternational Edition 41, 391-412; Hileman, R. E., et al. (1998)BioEssays 20, 156-167).

SUMMARY OF THE INVENTION

The present invention provides a heparin binding motif comprisingBZBXBX, wherein X represents any amino acid, Z represents an aromaticamino acid and B represents a basic amino acid.

of claim 1.

The present invention further provides a method for reducingcytotoxicity of eosinophil derived toxins comprising administering to asubject an effective amount of heparin, heparan sulfate, potentheparanase inhibitor or a polypeptide which has sequence as follows:BZBXBX, XBBBXXBX, XBBXBX, BBXXBBBXXBB, BXBBXB, XBBBXXBBBXXBBX, orTXXBXXTBXXXTBB, wherein X represents any amino acid, Z represents anaromatic amino acid, B represents a basic amino acid and T represents aturn

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The structure of synthetic heparin oligosaccharides used in thepresent invention. n=1-4 (n=1, dp3; n=2, dp5; n=3, dp7; n=4, dp9). FIG.2. FACE analysis to evaluate binding of ECP to various sugar-containingsubstrates. A, AMAC-labeled LMWH was incubated with or without ECP inPBS for 10 min at 25° C., and the binding reaction products wereseparated on a 1% agarose gel. The numbers above the gel indicate themolar ratio of ECP to LMWH. B, Representative oligosaccharide bindingpattern of ECP. AMAC-labeled heparin dp3 (10.8 nmol), dp5 (3.3 nmol),dp7 (1.4 nmol), dp9 (1.1 nmol), LMWH (0.03 nmol) and PI-88 (0.03 nmol)were incubated with or without 10-fold molar excess of ECP, and thebinding reaction products separated on a 1% agarose gel. C,Representative oligosaccharide binding pattern of EDN. AMAC-labeledheparin dp3 (10.8 nmol), dp5 (3.3 nmol), dp7 (1.4 nmol), dp9 (1.1 nmol),LMWH (0.03 nmol) and PI-88 (0.03 nmol) were incubated with or without10-fold molar excess of ECP, and the binding reaction products separatedon a 1% agarose gel.

FIG. 3. Heparin oligosaccharides inhibit ECP binding to cells. Beas-2Bcells were preincubated with heparin of different sizes in RPMI 1640medium for 30 min at 4° C. before incubation with MBP-ECP for anadditional 1 h. After treatment, the cells were washed with PBS andfixed with PFA. The level of bound MBP-ECP was assessed by ELISA. Theamount of MBP-ECP bound to cells without GAG treatment was set to 100%.C, control (cells were incubated with MBP.) The data shown are the meansof triplicate experiments.

FIG. 4. Identification of the heparin binding site in ECP. A, Alignmentof human and bovine RNases. The sequences between α2 and β1 of theRNases are aligned. Regions in other RNases that are highly similar tothe putative heparin binding site of ECP are boxed. Positively chargedamino acids are indicated in bold. B, Beas-2B cells were incubated withMBP-ECP for 1 h at 4° C. The amount of MBP-ECP bound to cells wasassessed as for FIG. 3.

FIG. 5. ITFE profiles of ECP wt and mt1 with dp5. Tryptophan titrationemission spectra of 0.2 mM ECP wt or mt1 bound with dp5. The emissionspectra at 340 nm were recorded. The resulting isotherms were fitted bynonlinear regression least-squares computer fit using the KaleidaGraphSynergy Software. ΔF, the relative fluorescence change, equalsF₀−F_(obs), where F_(o) and F_(obs) represent the initial and observedfluorescence values, respectively.

FIG. 6. CD spectra of ECP wt and mt1. The CD spectra were scanned from200 to 260 nm.

FIG. 7. Synthetic peptides inhibit ECP binding to cells. A, Beas-2Bcells were preincubated with peptides in RPMI 1640 medium for 30 min at4° C. before incubation with MBP-ECP for an additional 1 h. Aftertreatment, the cells were washed with PBS and fixed with PFA. The levelof bound MBP-ECP was assessed by ELISA. The amount of MBP-ECP bound tocells without peptide treatment was set to 100%. The data shown are themeans of triplicate experiments. B, Beas2-B cells were incubated withbiotinylated peptides for 1 h at 4° C. The amount of peptides bound tocells was assessed as for FIG. 3.

FIG. 8. Heparin-binding synthetic peptides. Human Beas-2B cells wereincubated with synthetic peptide (A, TAT; B, D1) at 4° C. for 30 min andthen washed, fixed and analyzed. Synthetic peptide was identified withmouse anti-biotin monoclonal antibody and FITC-conjugated goatanti-mouse antibody. The distribution of synthetic peptide was examinedby confocal microscopy. Scale bar, 10 mm.

FIG. 9. FACE analysis of synthetic peptides. AMAC-labeled LMWH wereincubated without or with increasing concentrations of C1, TAT, D1 andR1 peptides at room temperature for 15 min. Samples were loaded andseparated by agarose gel electrophoresis as described for FIG. 2.

FIG. 10. Titration profiles of synthetic peptides by synthetic heparinoligosaccharides. Titrations were carried out on 1 μM peptides in thepresence of heparin oligosaccharides. The K_(d) of eachpeptide-oligosaccharide interaction was determined by measuring thefluorescence change of intrinsic tryptophan at 340 nm. Data were fittedto a single-binding-site curve using nonlinear least-squares analysis.

FIG. 11. Isothermal titration calorimetry of heparin-peptideinteraction. The top panels show the differential power time course. Rawdata for sequential 6-μl injections of each synthetic peptide in PBSbuffer at 25° C., TAT (positive control), B, R1 (negative control), C,C1, and D, D1 into the sample cell containing 1.4 ml HMWH solution(10-20 μM). The total heat released in each injection is proportional tothe area under the corresponding peak. The lower panels show a fit ofthe integrated areas based on peptide binding. The solid line representsa non-linear least squares of the reaction heat for the injection.

FIG. 12. Cytotoxicity of wild-type and mutant ECP. Beas-2B cells wereincubated with increasing concentrations of wild-type and mt1 ECP at 37°C. for 48 h, followed by the MTT assay. The error bars show standarddeviation among triplicate experiments.

FIG. 13. Immunohistochemical localization of ECP using the SuperSensitive Non-Biotin HRP Detection System. Representativeimmunohistochemical staining patterns. ECP (red color) was detected intracheo-epithelial cells (arrow) and cartilage cells 1 hr post-ivinjection (A, B). Decreased ECP signal was observed intracheo-epithelial cells when ECP was co-injected with heparin (C). Acontrol section of lung tissue subjected to MBP injection was used as anegative control. Magnification: A, 200×; B, C, and D, 400×. Scale bars:20 μm.

DETAILED DESCRIPTION OF THE INVENTION

The term “eosinophil derived toxins” used herein means the toxinsderived from eosinophil and damage cells when the toxins are enteredinto the cells. The species of eosinophil derived toxins include but isnot limited to eosinophil cationic protein (ECP), eosinophil-derivedneurotoxin (EDN), eosinophil peroxidase (EPO, also called EPX), andmajor basic protein (MBP).

In the present invention, a linear heparin binding site on ECP and theshortest ECP-binding heparin oligosaccharide unit have been identified.Loop L3 of ECP mediates the interaction between ECP and cell surfaceHSPG, contributing to ECP cytotoxicity. Furthermore, the dissociationconstants between oligosaccharides and ECP were determined by tryptophanemission titration.

Accordingly, the present invention provides a heparin binding motifcomprising BZBXBX, wherein X represents any amino acid, Z represents anaromatic amino acid and B represents a basic amino acid. The presentinvention also provides a heparin binding motif of eosinophil cationicprotein comprising the above sequence. In the more embodiment of thepresent invention, the said motif is SEQ ID NO: 8.

The present invention further provides a method for reducingcytotoxicity of eosinophil derived toxins comprising administering to asubject an effective amount of heparin, heparan sulfate, potentheparanase inhibitor or a polypeptide which has sequence as follows:BZBXBX, XBBBXXBX, XBBXBX, BBXXBBBXXBB, BXBBXB, XBBBXXBBBXXBBX, orTXXBXXTBXXXTBB, wherein X represents any amino acid, Z represents anaromatic amino acid, B represents a basic amino acid and T represents aturn. The said subject is mammalian. The said polypeptide comprises ahigh portion of positively charged residues. In the embodiment of thepresent invention, the polypeptide comprises QRRCKN (SEQ ID NO:7) orRWRCKN (SEQ ID NO:8). In the more embodiment of the present invention,the polypeptide is SEQ ID NO: 3 (NYRWRCKNQNK) or SEQ ID NO: 4(NYQRRCKNQNK). The said heparin comprises low molecular weight heparin(LMWH, Sigma-Aldrish, average MW 3,000), high molecular weight heparin(HMWH, Sigma-Aldrish, average MW 16,000), heparan sulfate proteoglycans(HSPG) or synthetic heparin oligosaccharides and heparan sulfates fromthe degree of polymerization (dp) of 3 to 15. In the embodiment of thepresent invention, the synthetic heparan sulfates comprises degree ofpolymerization (dp) 5 to 9 (dp 3 to dp 9: MW 926-2,642; Molecularformula: P+nQ where P is a 2 methoxy-glucosamine, Q representsdisaccharide units consisted of hexuronic acid and glucosamine, and n isan integral. The molecular weight of X is 349 and that of Y ranges from333 to 573. Furthermore, the glucosamine of P could be substituted byother protecting group).

The cytotoxicity of eosinophil derived toxins is reduced by reducingendocytosis of eosinophil derived toxins such as ECP, EDN, EPO, and MBP.The method can further inhibit asthma. The said asthma includesECP/EDN-induced asthma, cytotoxic RNase-induced asthma and high pItoxin-induced asthma.

EXAMPLE Example 1 Materials

Mouse anti-MBP (maltose binding protein) was obtained from Santa CruzBiotechnology (Santa Cruz, Calif.). RNase A was purchased from Promega(Madison, Wis.). Chemicals were purchased from Sigma-Aldrich (St. Louis,Mo.) unless otherwise specified. Human ECP peptide NYRWRCKNQNK-biotin(C1, SEQ ID NO: 3), EDN peptide NYQRRCKNQNK-biotin (D1, SEQ ID NO: 4),RNasel peptide MTQGRCKPVNK-biotin (R1, SEQ ID NO: 5), and HIV-TATpeptide YGRKKRRQRRRK-biotin (TAT, SEQ ID NO: 6) were purchased fromGenemed Synthesis (South San Francisco, Calif.).

Example 2 Oligosaccharide Length-Dependence of ECP-Heparin Interaction

Recombinant hECP containing a C-terminal His₆ tag was expressed in E.coli BL21-CodonPlus(DE3) (Novagen, Madison, Wis.), purified bychromatography, and refolded as previously described (Boix, E., et al.(1999) Journal of Biological Chemistry 274, 15605-15614). MBP-ECP waspurified using amylase affinity chromatography.

The prior art demonstrated that binding and endocytosis of ECP are HSPGdependent (Fan, T. C., et al. (2007) Traffic 8, 1778-1795). An earlystudy demonstrated ECP purification using heparin-Sepharosechromatography (Gleich, G J., et al. (1986) Proc Natl Acad Sci USA 83,3146-3150). Thus, the minimal heparin length (or disaccharide unit)required to interact with ECP was investigated by FACE(Fluorescence-assisted carbohydrate electrophoresis).

Synthetic heparin oligosaccharide (FIG. 1) (Lee, C. J., et al. (2004) JAm Chem Soc 126, 476-477), low molecular weight heparin (LMWH) and PI-88(kindly provided by Progen Inc., Australia) were labeled with2-aminoacridone (AMAC) as described (Calabro, A., et al. (2000)Glycobiology 10, 273-281). AMAC-oligosaccharide and proteins wereincubated at room temperature for 15 min, loaded onto 1% gels andelectrophoresed as described (Holmes, O., et al. (2007) J Mol Biol 367,395-408).

Initially, ECP was co-incubated with LMWH at various molar ratios, andthe binding products were analyzed by gel electrophoresis. The decreasein the free LMWH signal was monitored as ECP concentration was increased(FIG. 2A). Subsequently, the dependence of ECP binding on heparinoligosaccharide size was examined in the presence of synthetic heparinoligosaccharides from the degree of polymerization (dp) 3 to 9 usingLMWH as a positive control. It was apparent that dp5 served as theshortest heparin fragment that retained the ability to bind ECP (FIG.2B). Furthermore, we tested a potent heparanase inhibitor (PI-88),undergoing clinical trials for its anti-angiogenic and anti-metastaticeffects (Joyce, J. A., et al. (2005) Oncogene 24, 4037-4051; Parish, C.R., et al. (1999) Cancer research 59, 3433-3441), that contains amixture of highly sulfated mannose-containing di- to hexasaccharides(Yu, G., et al. (2002) European journal of medicinal chemistry 37,783-791; Ferro, V, Li, C., et al. (2002) Carbohydrate research 337,139-146). Interestingly, this heparin mimetic could also bind ECP (FIG.2B). In addition, similar results were observed for EDN (FIG. 2C).

Example 3 Competitive Inhibition of ECP Binding to Cells by SyntheticOligosaccharides

Beas-2B, a human bronchial epithelial cell line, was cultured in RPMI1640 medium (Sigma-Aldrich) supplemented with heat-inactivated 10% fetalbovine serum (Gibco/Invitrogen, Carlsbad, Calif.).

The ability of MBP-ECP to bind cells in the presence of serial dilutionsof oligosaccharides or peptides was determined as described (Fan, T. C.,et al. (2007) Traffic 8, 1778-1795). At the present invention, syntheticheparins (dp3-9) were tested for their ability to interfere with ECPbinding to Beas-2B cells. Briefly, confluent monolayers of Beas-2B cellsin 96-well plates were pretreated with various concentrations ofoligosaccharides or peptides in serum-free RPMI 1640 medium at 4° C. for30 min before incubation with 5 μg/ml MBP-ECP at 4° C. for 1 h. Thecells were then washed with ice-cold PBS and fixed with 2% PFA at roomtemperature for 15 min prior to blocking with 2% BSA/PBS at roomtemperature for 90 min. The level of bound MBP-ECP was quantified byELISA analysis. MBP-ECP was detected using mouse monoclonal anti-MBP andgoat anti-mouse horseradish peroxidase (HRP)-conjugated secondaryantibody, followed by the enhanced chemiluminescence detection system.The amount of MBP-ECP bound to cells without oligosaccharide or peptidetreatment was set to 100%.

Beas-2B cells were preincubated with oligosaccharides, and bound ECP wasdetected essentially as described (Fan, T. C., et al. (2007) Traffic 8,1778-1795). The degree of inhibition increased with increasingoligosaccharide length (FIG. 3). Fifty micrograms per mililiter ofheparin dp5 inhibited 50% of ECP binding to cells, and the same amountof dp7 and dp9 was capable of inhibiting over 70% and 80% of ECPbinding, respectively. These data revealed that pentasaccharide was theminimal length sufficient to interfere with ECP binding to Beas-2Bcells. In addition, the concentration dependence of PI-88 againstcellular binding of ECP was similar to that of dp7 (FIG. 3).

Example 4 Identification of Heparin-Binding Sequence in ECP

A consensus sequence of the heparin binding site (e.g., XBBXBX orXBBBXXBX) has been found in many GAG-binding proteins or peptides(Hileman, R. E., et al. (1998) BioEssays 20, 156-167; Cardin, A. D. andWeintraub, H. J. (1989) Arteriosclerosis Dallas, Tex. 9, 21-32).Inspection of the sequences of human RNase A family members revealed asurface loop L3 region, ³⁴QRRCKN (SEQ ID NO: 7), in EDN that exactlymatches the XBBXBX motif (FIG. 4A), but no consensus heparin-bindingmotif was found in ECP. Therefore, it was speculated that residues 34-39(34RWRCKN, SEQ ID NO: 8) in ECP that correspond to the consensus motifin EDN might also bind heparin. To determine whether this regioncontributes to cellular binding, cell ELISA analysis was conducted usingMBP-ECP and MBP-ECP mt1 containing the mutations R34A/W35A/R36A/K38A.

Amino acid residues R34, W35, R36, and K38 of ECP were simultaneouslysubstituted to alanine using QuickChange site-directed mutagenesis(Stratagene, La Jolla, Calif.) and the resultant mutant was named ECPmt1. The primers used were as follows: mt1 forward,5′-TATGCAGCGGCTTGCGCAAACCAAAAT-3′ (SEQ ID NO: 1), and mt1 reverse,5′-TTTGCGCAAGCCGCTGCATAATTGTTA-3′ (SEQ ID NO: 2). E. coliBL21-CodonPlus(DE3) cells were used to transform various plasmids. Thismutant had only 50% of the cell-binding activity, indicating theimportance of the ³⁴RWRCKN motif in ECP for cellular HS binding (FIG.4B).

Example 5 Characterization of Association Between ECP and HeparinOligosaccharides

The binding affinities of wild-type and mutant ECP for heparinoligosaccharides were subsequently monitored by intrinsic tryptophanfluorescence titration (Lau, E. K., et al. (2004) The Journal ofbiological chemistry 279, 22294-22305).

Binding of ECP to heparin was monitored by changes in intrinsictryptophan fluorescence emission (IFTE). ECP (200 nM) in PBS at 25° C.was titrated with small aliquots of a high concentration ofpentasaccharides with minimal dilution (<2%). Protein fluorescencemeasurements were recorded 2 min after each addition on a Hitachi 8000spectrofluorimeter at emission wavelength of 340 nm using an excitationwavelength of 280 nm. AF, the relative fluorescence change, equalsF₀−F_(obs), where F₀ and F_(obs) represent the initial and observedfluorescence values, respectively. Binding constants were estimated fromthe titration data using a nonlinear least-squares computer fit to theequation based on 1:1 binding stoichiometry (Venge, P. and Bystrom, J.(1998) The international journal of biochemistry & cell biology 30,433-437):

ΔF=ΔF_(max)×([P]+[H]+K_(d)−(([P]+[H]+K_(d))²−4×[P]×[H])^(1/2))/(2×[P])(Eq. 1) where ΔF_(max) is the maximum relative fluorescence change, P isthe total concentration of ECP, H is the concentration of heparin, andK_(d) is the dissociation constant for the ECP-heparin interaction.

The change in tryptophan fluorescence revealed that wild-type ECP boundto dp5 with high affinity, and the corresponding K_(d) was 139.6 nM(FIG. 5). As expected, a 4- to 5-fold increase in K_(d) to 568.1 nM wasobserved for ECP mt1 (R34A/W35A/R36A/K38A) (Table I), indicatingdecreased heparin binding activity

TABLE I Dissociation constant (K_(d)) determination for wild-type ECPand mt1 with heparin oligosaccharides. ECP wt ECP mt1 OligosaccharideK_(d) (nM) K_(d) (nM) dp9 62.6 400.3 dp5 139.6 568.1 K_(d) was measuredin PBS at 25° C. The change in intrinsic tryptophan fluorescence was fitby the equation 1 to obtain K_(d).

Circular dichroism spectroscopy (CD spectroscopy) was used to comparethe conformations of wild-type ECP and ECP mt1. CD spectra were recordedusing an Aviv model 202 CD Spectrometer equipped with a 450-watt Xenonarc lamp. Far-UV spectra were recorded at 25° C. from 200 to 250 nmusing a 0.1-cm cuvette containing 10 μM protein in PBS. The CD spectrawere recorded at 1.5-min intervals with a bandwidth of 1 nm. Eachspectrum is an average of three consecutive scans and was corrected bysubtracting the buffer spectrum. The results showed that the secondarystructure of ECP mt1 was very similar to that of wild-type ECP (FIG. 6).These results strongly indicated that the decrease in heparin bindingaffinity resulted from a loss of the specific recognition sequencemotif, and not a conformational change in ECP mt1.

Example 6 Competitive Inhibition of ECP Binding to Cells by SyntheticPeptides

To investigate whether the RWRCK (SEQ ID NO: 8) motif is directlyresponsible for heparin binding, cell ELISA was conducted using asynthetic peptide, C1 (SEQ ID NO: 3), along with a positive controlpeptide derived from HIV-TAT, TAT (SEQ ID NO: 6) (Vives, E., et al.(1997) The Journal of biological chemistry 272, 16010-16017; Ziegler, A.and Seelig, J. (2004) Biophysical journal 86, 254-263). Thedose-dependent competition of these peptides is shown in FIG. 7A.Binding of MBP-ECP to cell-surface HS was significantly reduced in thepresence of both C1 (SEQ ID NO: 3) and TAT (SEQ ID NO: 6) peptides.Therefore, synthetic peptides with heparin binding activity may competewith ECP for cellular binding. Furthermore, the cell surface bindingability of peptides was tested using cell ELISA assay. As expected,similar to the TAT (SEQ ID NO: 6), C1 (SEQ ID NO: 3), and D1 (SEQ ID NO:4) peptides bound to the cell surface, whereas R1 (SEQ ID NO: 5) peptidewas devoid of such function (FIG. 7B).

Example 7 Interaction Between Synthetic Heparin Binding Peptide and CellSurface

To investigate whether the heparin binding QRRCK motif on EDN,corresponding to the “RWRCK” motif on ECP (SEQ ID NO: 8), was directlyresponsible for heparan sulfate binding on cell surface, Beas-2Bcell-peptide binding was conducted using a synthetic peptide, D1 (SEQ IDNO: 4), and a positive control, TAT (SEQ ID NO: 6) purchased fromGenemed Synthesis (South San Francisco, Calif.). The data demonstratedthat synthetic peptide D1 (SEQ ID NO: 4) bound to Beas-2B cell surfaceheparan sulfate at low temperature, in consistent with the resultsobtained from cell-ELISA competitive inhibition experiments shown inFIG. 8.

Example 8 Interaction of Synthetic Peptides with LMWH

The ability of C1 (SEQ ID NO: 3) to directly interact with LMWH wasfurther investigated by FACE analysis. As expected, a decreased amountof free AMAC-LMWH signal was observed with increasing concentration ofC1 (SEQ ID NO: 3) peptide or TAT (SEQ ID NO: 6) peptide (FIG. 9). Inaddition, because EDN contains a conventional heparin binding sequence,the corresponding peptide, D1 (SEQ ID NO: 4), was also synthesized andtested. As shown in FIG. 8, D1 (SEQ ID NO: 4) bound heparin as tightlyas C1 (SEQ ID NO: 3). Interestingly, although the peptide segment in thecorresponding location of human RNase 1 (R1, SEQ ID NO: 5) also containsseveral positively charged residues, it did not bind heparin. Takentogether, these results indicate that the RWRCK (SEQ ID NO: 8) motifwithin loop L3 of ECP serves as a specific heparin binding site.

Example 9 Characterization of Association Between C1 and HeparinOligosaccharides

The definitive heparin binding activity of the C1 (SEQ ID NO: 3) peptidewas determined by ITFE, which clearly demonstrated that C1 has highaffinity for heparin (FIG. 10). The K_(d) values obtained for LMWH, dp9,and dp5 binding to the C1 (SEQ ID NO: 3) peptide were 192.0, 229.1, and274.8 nM, respectively (Table II), indicating that C1 (SEQ ID NO: 3)bound tighter to longer heparin oligosaccharides.

TABLE II Determination of dissociation constants (K_(d)) for C1 peptidewith heparin oligosaccharides. Oligosaccharide K_(d) (nM) LMWH 192.0 dp9229.1 dp5 274.8

Example 10 Characterization of Association Between Synthetic HeparinBinding Peptides and HMWH

In general exothermal reaction takes place upon molecular bindingbetween synthetic peptide and HMWH. As expected, the positive controlTAT (SEQ ID NO: 6) peptide showed significant exothermal reaction modeemploying ITC (FIG. 11A), whereas the negative control R1 (SEQ ID NO: 5)peptide revealed no heat release (FIG. 11B), strongly suggesting that nobinding occurred between the test molecules. As for the synthetic C1(SEQ ID NO: 3) and D1 (SEQ ID NO: 4) peptides, exothermal reaction modeswere clearly observed (FIGS. 11C-D). These data further proved thatpeptide motifs C1 (SEQ ID NO: 3) and D1 (SEQ ID NO: 4) acted as crucialin vitro HMWH binding sites residing in human eosinophil RNases.

Example 11 Growth Inhibitory Effect of ECP

The inhibition of lymphocyte and mammalian cell growth by ECP has beenreported (Fan, T. C., et al. (2007) Traffic 8, 1778-1795; Maeda, T., etal. (2002) European Journal of Biochemistry 269, 307-316). As aphysiological test, the cytotoxicity of ECP mt1 was monitored by MTTassay. The effect of ECP on the cell growth was determined by acolorimetric assay using3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (USBiological, MA, USA). Cells were plated in a 96-well plate (5000cells/well) and incubated at 37° C. overnight. Each sample was incubatedwith the indicated concentration (1-100 μM) of ECP. Forty-eight hoursafter treatment with ECP, MTT was added, and cell growth was monitoredat A₅₇₀ to measure the mitochondrial-dependent formation of a coloredproduct.

As compared with wild-type ECP, ECP mt1 (R34A1W35A/R36A/K38A) exhibiteda 2- to 3-fold increased IC₅₀ value toward the Beas-2B cell line (FIG.12, Table III). Thus, ECP mt1 without the key heparin binding sequencemotif appears to be less cytotoxic than wild-type ECP, presumably due toa lesser interaction with the cell surface, which in turn leads to lessendocytosis.

Table III. IC₅₀ Values for Cytotoxic ECP.

TABLE III IC₅₀ values for cytotoxic ECP. Protein IC₅₀ (μM) ECP wt^(a)16.05 ECP mt1 38.52 ^(a)Data taken from reference. (Fan, T. C., et al.(2007) Traffic 8, 1778-1795)

Example 12 Immunohistochemical Localization of ECP

To better understand the possible cellular targets of ECP, recombinantmature ECP was injected into the rat circulation through the tail vein.

Adult specific-pathogen-free (SPF) Sprague-Dawley rats (Narl:SD) withbody weight (BW) of 200-300 g, were purchased and maintained at theNational Laboratory Animal Center (NLAC) in Taiwan. The rats wereseparated into three groups. In group 1, each rat was injected with 5nmol of ECP through the tail vein. In group 2, each rat was co-injectedwith heparin (FRAGMIN®, average MW 6000, 5000 IU/0.2 ml) and 5 nmol ofECP. In group 3, each rat was injected with 5 nmol of MBP as thenegative control. All animals were sacrificed using CO₂ narcosis 1 hafter the injection of these agents. The lung and trachea of these ratswere taken and immediately fixed with 10% neutral buffered formaldehyde.The tissue samples were processed by routine methods to prepare paraffinwax-embedded block. These blocks were then sectioned into 6-μm slices.All tissue sections were examined using the Super Sensitive Non-BiotinHRP Detection System (BioGenex Laboratories, San Ramon, Calif.) asdescribed (Liang, C. T., et al. (2007) Journal of comparative pathology136, 57-64). Briefly, the mouse anti-ECP or anti-MBP monoclonal antibodywas used as the primary antibody. Antigen unmasking was performed byimmersion of sections in 5% Trilogy (Cell Marque, Rocklin, Calif.)antigen unmasking solution in Milli-Q water and boiled at 121° C.Endogenous peroxidase activity was quenched with hydrogen peroxide (3%)in methanol. These sections were then incubated in Power Block solution,and mouse anti-ECP or anti-MBP at 1/200 dilution was applied and leftfor 24 h. The sections were incubated with Super Enhancer reagent,followed by Polymer-HRP reagent, and then incubated with 3-amino, 9ethyl-carbazole chromogen solution. The sections were finallycounterstained with Mayer's hematoxylin and mounted with Super Mountpermanent aqueous mounting media prior to examination with a lightmicroscope (Zeiss—Axioplan, Germany).

Immunohistochemical localization showed strong internalization of ECP inthe tracheo- and broncho-epithelial cells 1 h post-injection (FIGS.13A-B). ECP internalizaiton was reduced when co-injection with heparinwas carried out (FIG. 13C). For the negative control, no MBP signal wasdetected in tracheo- and broncho-epithelial cells, despite the MBPsignal that could be detected in circulating blood (FIG. 13D).

1. An isolated peptide consisting of the amino acid sequence set forthin SEQ ID NO:
 8. 2. The isolated peptide as recited in claim 1, whereinthe isolated peptide is a heparin binding site of eosinophil cationicprotein.
 3. The isolated peptide as recited in claim 1, which hasheparin or heparan sulfate binding activity.